Multicellular organisms utilize a variety of mechanisms to defend against microbial invasion. These include anatomical and chemical barriers, as well as numerous cell-mediated and humoral responses. Collectively these defenses aim to eliminate offending microorganisms. Epithelial surfaces of many tissues are continually exposed to potential pathogenic organisms, yet the incidence of infectious disease following these encounters is relatively small, highlighting the effectiveness of defense mechanisms at these sights. Manifestations of abnormality of these defenses in the intestinal tract may include various forms of diarrhea associated with pathogenic bacteria and ulcerative diseases including inflammatory bowel disease, necrotizing enterocolitis and gastric ulcer disease.
Peptide based antimicrobial defense is a conserved component of host defense, and is found in both the animal and plant kingdoms (for reviews see Boman and Hultmark, Ann Rev. Microbiol., 41: 103-126 (1987); Bevins and Zasloff, Ann Rev. Biochem, 59:395-414 (1990); Spitznagel, J Clin. Invest. 86: 1381-86 (1990); Boman, Cell 65: 205-207 (1991); Lehrer et al., Cell 64: 229-230 (1991)). The size and structure of the antimicrobial peptides shows significant diversity, but in general, they are membrane-active amphipathic molecules with a net positive charge at neutral pH. There are two broadly defined families of these cationic peptides: linear peptides (for example cecropins; Steiner, et al., Nature 292: 246-248 (1981); and magainins; (Zasloff, Proc. Natl. Acad. Sci. USA 84: 5449-5453 (1987)) and cystsine-rich peptides. The latter include mammalian defensins (Ganz et al., Eur J Haematol 44: 1-8 (1990)), tracheal antimicrobial peptide (Diamond, et al., Proc Natl Acad Sci (USA) 88:3952-3956 (1991), bovine bactenecins, (Romeo et al. J Biol Chem 263: 9573-9575 (1988)), insect royalisin, (Fujiwara et al., J Biol Chem 265:11333-11337 (1990)), tachyplesins (Nakamura, et al., J Biol Chem 263:16709-16713 (1988); Shigenaga, et al., J. Biol Chem 265: 21350-21354 (1990), and plant thionins (Olson and Samuelsson, Acta Chem Scand 26: 585-595 (1972); Ozaki, et al., J. Biochem 87:549-555 (1980); Bohlmann and Apel, Mol Gen Genetics 207: 446-454 (1987); Bohlmann, et al., EMBO J 7: 1559-1565 (1988).
Defensins are cysteine-rich basic peptides which have been isolated from myeloid-derived cells of several mammalian species (For recent reviews see Ganz, et al., Eur J Haematol 44:1-8 (1990); Lehrer, et al., Cell 64: 229-230 (1991). Defensins have in vitro antimicrobial activity against bacteria; Selsted, et al., Infect Immun 45: 150 (1984); Ganz, et al., J Clin Invest 76: 1427-1435 (1985); fungi; Ganz, et al., J Clin Invest 76: 1427-1435 (1985); Borenstein, et al., Infect Immun 59: 1359-67 (1991); and enveloped viruses (Lehrer et al., J Virol 54: 467 (1985); Daher, et al., J Virol 60: 1068-1074 (1986). Defensins are characterized by eleven conserved residues within the sequence, including six cysteines which participate in intramolecular disulfide bonds (Selsted and Harwig, J Biol Chem 264:4003-4007 (1989). This disulfide array is important for structure and activity of defensins. Evidence suggests that their antimicrobial activity is a direct result of their ability to selectively disrupt membranes (Lehrer, et al., J Clin Invest 84:553-561 (1989)); Lichtenstein, J Clin Invest 88: 93-100 (1991), possibly by channel formation (Kagan, et al, Proc Natl Acad Sci (USA) 87: 210-214 (1990)). The high-resolution crystal structure of human defensin-3 has recently been determined (Hill, et al. Science 251: 1481-1485 (1991), and suggests several specific models for the interaction of defensins with lipid membranes, the site of defensin antimicrobial activity. In addition to antimicrobial activity, certain defensins have other biological activities including, monocyte chemotaxis (Territo, et al., J Clin Invest 84: 2017-2020 (1989), adrenocortical suppression (Singh et al., Bioch Biophys Res Commun 155: 524-529 (1988), nifedipine-sensitive calcium channel activation (MacLeod et al., Proc Natl Acad Sci 88: 552-556 (1991) and eucaryotic cell cytotoxicity (Okrent et al., Am Rev Respir Dis 141: 179-185 (1990). In addition to myeloid expression, recent investigations in the mouse (Ouellette and Cordell, Gastroenterol 94: 114-121 (1988), Ouellette et al., J Cell Biol 108: 1687-1695 (1989), Ouellette and Lualdi, J Biol Chem 265: 9831-9837 (1990) and in the cow (Diamond and Bevins, (In preparation) (1991)), Diamond et al., Proc Natl Acad Sci (USA) 88:3952-3956 (1991) show that the defensin-related peptides, cryptdin and tracheal antimicrobial peptide, are also expressed in epithelial tissues.
In humans, defensins are major constituents of the azurophilic granules of neutrophils (Ganz, et al., J Clin Invest 76: 1427-1435 (1985), Selsted, J Clin Invest 76: 1436-1439 (1985); Rice, Blood 70: 757-765 (1987), Lehrer, Hematol Oncol Clin North Am 2: 159-169 (1988) and are though to contribute to the non-oxidative killing of microorganisms by these circulating leukocytes. (Lehrer et al., Hematol Oncol Clin North Am 2: 159-169 (1988). Defensins and other proteins of the azurophilic granules have been shown to enter the phagolysosome vesicles of neutrophils during phagocytosis of bacteria (Joiner et al., J Cell Biol 109: 2771-2782 (1989). Four myeloid-derived human defensins have been isolated and characterized (Ganz et al., J Clin Invest 76: 1427-1435 (1985); Selsted, et al., J Clin Invest 76:1436-1439 (1985); Singh et al, Bioch Biophys Res Commun 155: 524-529 (1988), Gabay et al., J Immunol 143:1358-1365 (1989), Wilde, J Biol Chem 264:11200-11203 (1989). Human defensins 1 and 3 are 30 amino acid peptides, differing in sequence by only a single residue at their amino terminus. (Selsted, et al., J Clin Invest 76:1436-1439 (1985). The cloned cDNAs for these two defensins (Daher, et al., Proc Natl Acad Sci USA 85:7327-7331 (1988); Mars et al., Blood 71: 1713-1719 (1988); Wiedemann et al., Leukemia 3: 227-234 (1989) are greater than 98% identical in nucleotide sequence, with a single nucleotide difference in codon 65 of the putative prepropeptides accounting for the alanine or aspartic acid residues in the mature defensins 1 and 3 respectively. Defensin 2, a 29 amino acid peptide, is identical to defensins 1 and 3 except that it lacks either of these amino acids at its amino-terminus (Selsted, et al., J Clin Invest 76: 1436-1439 (1985). The cDNA for this defensin has not yet been cloned, and it is not clear if it is a product of a distinct gene, or a post-translational proteolytic modification of defensins 1 or 3. Defensin 4 is quite different from other human defensins in primary structure. This 33 residue peptide essentially shares only the consensus residues that characterize defensins (Singh et al., Bioch Biophys Res Commun 155: 524-529 (1988); Wilde, et al., J Biol Chem 264; 11200-11203 (1989), and neither its cDNA or gene have been described. By in situ hybridization histochemistry, defensin cDNA probes detect a message expressed in a relatively narrow window of granulocyte development. The mRNA is abundant in late promyelocytes and early myelocytes, precursors of the mature circulating neutrophils (Mars et al., Leukemia 1: 167-172 (1987), Wiedemann, et al., Leukemia 3: 227-234 (1989), as well as other granular leukocytes (Mars, et al. Leukemia 1: 167-172 (1987) of human bone marrow, but is undetectable by northern blot analysis in circulating neutrophils (Daher, et al., Proc Natl Acad Sci USA 85:7327-7331 (1988).
Novel defensin peptides having antimicrobial and anti-inflammatory activity are greatly desired. Defensin peptides particularly suitable for use in the gastrointestinal tract of humans are particularly desireable since they may be effective for treatment of gastrointestinal conditions such as various forms of diarrhea associated with pathogenic bacteria and ulcerative diseases including inflammatory bowel syndrome, necrotizing enterocolitis and gastric ulcer disease.